Cortical activity in the intact brain is characterized by intermittent sustained membrane depolarizations called "up-states". The task-specific computations of a given cortical region are superimposed on top of this heightened activity. In the case of the prefrontal cortex (PFC), recurrent excitation of deep layer neurons is thought to underlie sustained activity during working memory. Dopamine (DA) modulates working memory, however, it has not been feasible to investigate its detailed biophysical effects in the awake animal. Instead, most of our knowledge about dopamine's effects on membrane excitability or synaptic transmission derives from recordings in "quiet" acute brain slice preparations. However, up-states and sustained activity are a network phenomenon and the asynchronous firing that occurs across neurons results from the interplay between intrinsic excitability and recurrent synaptic transmission. The present proposal investigates DA modulation of active cortex using an organotypic co-culture preparation of the PFC, hippocampus and ventral tegmental area (VTA). These cultures exhibit sustained activity (up-states) and possess key inputs required for working memory, namely the hippocampus and the DA input from the VTA. The present proposal tests two hypotheses generated from in vitro brain slice preparations and computational modeling studies. The first hypothesis posits that sustained activity during up-states can be modulated by DA in a dose-dependent manner. Experiments in Specific Aim 1 will use whole-cell current clamp recordings to test whether there is an "optimal" range of DA modulation in which action-potential firing and up-state duration are enhanced, while outside of this optimal range sustained activity will be reduced. Control experiments will assess the contribution of tonic vs. phasic release of DA from the VTA, and will test whether DA has network specific effects. The concentration of DA in the slice will be monitored simultaneously via fast-scan cyclic voltammetry. The second hypothesis states that DA acts on GABA and NMDA currents to modulate persistent activity during up-states. Combined voltage-clamp and calcium imaging experiments will be used to measure the modulation of GABA and NMDA currents by a high dose or low dose of DA, respectively. These studies will provide insights into the architecture of sustained activity and DA modulation of active PFC networks; information critical for our understanding of the normal and pathological conditions of the PFC. [unreadable] [unreadable] [unreadable]